Influenza virus-like particle (vlp) compositions

ABSTRACT

Influenza virus-like particles (VLPs) comprising influenza antigenic polypeptides are described. Also described are compositions comprising these VLPs as well as methods of making and using these VLPs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/796,988, filed Apr.30, 2007, which claims the benefit from U.S. Provisional ApplicationNos. 60/798,363, filed May 5, 2006; 60/796,735, filed May 1, 2006; and60/796,799, filed May 1, 2006. The entire disclosures of theabove-referenced applications are incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funds used to support some of the studies disclosed herein were providedby grant number 1 R43 AI063830-01 awarded by the National Institute ofAllergy and Infectious Disease (NIAID) of the National Institutes ofHealth (NIH). The U.S. Government may have certain rights in theinvention.

TECHNICAL FIELD

Virus-like particles (VLPs) containing influenza antigens are described,as are methods and making and using these VLPs.

BACKGROUND

The influenza A virus is a well characterized virus that infects humansas well as a large number of other species. See, e.g., U.S. PatentPublication No. 20050186621. All of the sixteen subtypes of influenza Avirus circulate in wild birds and domestic avian species. Few influenzasubtypes are epidemic among humans, but periodically pandemic strainsderived from animals or birds unpredictably emerge causing wide spreaddisease of high morbidity and mortality.

The influenza A virus (H1N1) 1918 was the most virulent and highlycontagious airborne pathogen in the recorded history of human infectiousdiseases. After the 1918 pandemic, this virus gradually changed to aless virulent strain and with time the original highly pathogenic virusno longer circulated in humans. However, given the vast reservoir ofinfluenza viruses in wild bird populations as well as the complexepidemiology, biology and evolutionary characteristics of this virus itis possible that a similar or related strain will reemerge creating aserious threat to global public health. In addition, the recentre-creation of this pathogen raises serious concern that the availabletechnology could lead to reconstruction of this virus as a biologicalweapon.

Avian influenza viruses are also a constant threat to humans because ofcross-species transmission which allows for their adaptation to humansthrough mutations or reassortment with the potentially of causing aglobal pandemic. Avian to human transmission has resulted in more than300 human cases of avian influenza worldwide with a mortality rate closeto 40%. See, Thomas & Noppenberger (2007) Am J Health Syst Pharm.64(2):149-65, Epidemiology of WHO-confirmed human cases of avianinfluenza A(H5N1) infection. Wkly Epidemiol Rec. 2006 Jun. 30;81(26):249-57. Indeed, since 1997, the avian influenza virus H5N1 hasbeen causing massive disease outbreak in domestic poultry as well as inother avian and mammalian species. In addition, a recent outbreak of thehighly pathogenic avian influenza subtype H7N7 in the Netherlandsresulted in multiple human infections, one of which proved fatal.

Traditionally, influenza vaccines are produced in fertilized chickeneggs. Eleven days after fertilization, a single strain of influenzavirus is injected into the eggs. The virus multiplies in the infectedembryo and after several days of incubation, the eggs are opened thevirus harvested with the fluid surrounding the embryo, purified,chemically inactivated and combined with other similarly producedstrains to generate an influenza vaccine. On average, one to two eggsare needed to produce one dose of vaccine and the entire productionprocess lasts at least six months. Given the long production times, itis unlikely that egg-based production of flu vaccines could be used tocontain a flu pandemic.

Therefore, there remains a need for compositions and methods thatprevent and/or treat infection with the various highly virulent andtransmissible influenza strains.

SUMMARY

In one aspect, a VLP comprising at least one influenza matrix protein(M1 and/or M2), a first influenza HA protein and a first influenza NAprotein, wherein the first influenza HA is selected from the groupconsisting of HA1, HA5 and HA7 and the first NA protein is selected fromthe group consisting of NA1 and NA7. In certain embodiments, the VLPincludes a single matrix protein, for example M1. In other embodiments,the VLP comprises M1 and M2. In certain embodiments, the VLP comprisesHA1 and NA1. In other embodiments, the VLP comprises HA7 and NA7. Instill further embodiments, the VLP comprises HA5 and NA1.

Any of the VLPs described herein may further comprise an influenzanucleoprotein (NP) and/or one or two proteins of the polymerase complex(made up of PB1, PB2 and PA). For example, the VLP may include NP and/orPB1, PB2 and PA; NP and/or PB1 and PB2; NP and/or PB1 and PA; and/or NPand/or PB2 and PA.

Any of the VLPs described herein may comprise chimeric (hybrid)influenza proteins (HA, NA, M1 and/or M2). In certain embodiments, theHA and/or NA proteins are chimeric. For example, in certain embodiments,a portion of the first influenza HA protein is replaced with ahomologous region from a second influenza HA protein, wherein the secondinfluenza HA protein is derived from a different strain than the firstinfluenza HA protein; and/or a portion of the first NA protein isreplaced with a homologous region from second influenza NA protein,wherein the second influenza NA protein derived from a different strainthan the first influenza NA protein. The transmembrane and/orcytoplasmic domains may be replaced with a homologous region from adifferent influenza protein (e.g., influenza virus A/PR/8/34 orA/Udorn/72). In certain embodiments, the transmembrane domain of aglycoprotein is replaced. In other embodiments, the cytoplasmic tailregion of a glycoprotein is replaced. In yet other embodiments, thetransmembrane domain and the cytoplasmic tail region of one or moreglycoproteins are replaced with domains from different influenzaproteins.

In another aspect, described herein is a host cell comprising any of theVLPs as described above. The host cell may be an insect, plant,mammalian, bacterial or fungal cell.

In yet another aspect, a cell stably transfected with a sequenceencoding at least one influenza matrix protein or an influenzaglycoprotein is provided. The cell may be an insect, plant, mammalian,bacterial or fungal cell. In certain embodiments, the cell is amammalian cell line.

In another aspect, a packaging cell line is provided for producinginfluenza VLPs as described herein. The cell line is stably transfectedwith a sequence encoding at least one of the influenza proteins (matrixand glycoprotein) forming a VLP as described herein. Sequences encodingthe remaining VLP-forming influenza proteins are introduced into thepackaging cell line under conditions such that VLPs are formed. Incertain embodiments, sequences encoding at least one influenza matrixprotein (M1 and/or M2) are stably integrated into the packaging cellline and sequences encoding the glycoproteins expressed on the surfaceof the VLP are introduced into the cell such that the VLP is formed. Inother embodiments, sequences encoding one or more of the glycoproteinsare stably integrated into the cell to form a packaging cell line andVLPs are formed upon introduction of sequences encoding M1 and,optionally, M2. The packaging cell may be an insect, plant, mammalian,bacterial or fungal cell. In certain embodiments, the packaging cell isa mammalian cell line.

In yet another aspect, the disclosure provides an immunogeniccomposition comprising any of the VLPs described herein and apharmaceutically acceptable excipient. In certain embodiments, thecompositions further comprise one or more adjuvants.

In a still further aspect, a method of producing a VLP as describedherein is provided, the method comprising the steps of: expressing oneor more polynucleotides encoding the M1, HA and NA (and optionally M2)proteins in a suitable host cell under conditions such that the VLPsassemble in the host cell; and isolating the assembled VLPs from thehost cell. The host cell can be a mammalian cell, an insect cell, ayeast cell or a fungal cell. In certain embodiments, an expressionvector comprising one or more polynucleotides operably linked to controlelements compatible with expression in the selected host cell areintroduced into the host cell. The expression vector may be a plasmid, aviral vector, a baculovirus vector or a non-viral vector. In certainembodiments, one or more of the polynucleotides are stably integratedinto the host cell. Alternatively, one or more of the polynucleotidesmay be transiently introduced into the host cell.

In another aspect, provided herein is a method of generating an immuneresponse in a subject to one or more influenza viruses, the methodcomprising the step of administering a composition comprising one ormore VLPs as described herein to the subject. In certain embodiments,the composition is administered intranasally. Any of the methods mayinvolve multiple administrations (e.g., a multiple dose schedule).Furthermore, any of the methods described herein may generate an immuneresponse to more than one influenza virus strain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing of the position and orientation ofthe four influenza genes contained in the quadruple baculovirusrecombinant construct. Expression of the HA and NA of the 1918 influenzaA virus together with the M1 and M2 proteins derived from the influenzavirus A/Udorn/73 results in VLP formation. The transmembrane domains andcytoplasmic tails of the 1918 HA and NA were replaced by those of theinfluenza A/Udorn virus glycoproteins (NH2 terminal in NA and COOHterminal in HA). In this construct, the HA and M1 genes are in oppositeorientation to each other and under the transcriptional control of thebaculovirus polyhedrin promoter whereas the M2 and NA are under thetranscriptional control of the p10 promoter and in opposite orientationto each other.

FIG. 2, panels A, B and C, show production of 1918 VLPs by infected Sf9cells as monitored by expression of the green fluorescence protein (GFP)at day 1 (panel A), day 2 (panel B), and day 3 (panel C), postinfection. The quadruple baculovirus recombinant expresses GFP inaddition to the four influenza proteins required for VLP assembly;therefore GFP expression served as an indicator of infection and VLPprotein production.

FIG. 3, panels A and B, depict Western blot analysis of gradientpurified 1918 VLP vaccine. The top two fractions of the gradient werecollected and analyzed by Western blot using as primary antibodies ananti-1918 HA (panel A) mouse monoclonal and an anti-M1 (panel B) mousepolyclonal (Serotec, N.Y.). Both proteins were detected in the purifiedVLP vaccine fractions.

FIG. 4 shows that the 1918 VLPs agglutinate turkey red blood cells (RBC)but not chicken red blood cells, as evaluated in a standardhemagglutination assay.

FIG. 5 is a graph depicting neuraminidase activity (expressed as theoptical density (OD) at wavelength 549 nm) of purified VLPs as assayedin an enzymatic reaction using fetuin as a substrate.

FIG. 6 is a graph depicting serum antibody responses to 1918 virus-likeparticles (VLPs). Light gray bars depict detergent solubilized virusELISA while dark gray bars depict rHA 1918 cell based ELISA. Datarepresents the average of all the individual measurements.

FIG. 7 is a graph depicting body weight of groups of mice followingintranasal influenza challenge and presented as group weight average ingrams versus days post infection Animals who received VLP alone areshown on the line joining black squares; animals who received VLP andCpG are shown on the line joining black diamonds; animals receivinginactivated virus vaccine are shown on the line joining black triangles;and control animals (no vaccine) are shown on the line joining “x.”

FIG. 8 is a graph depicting virus titers in nasal tissue of micereceiving VLP vaccine alone (line joining black diamonds); VLP and CpG(line joining black squares); inactivated virus vaccine (line joiningblack triangles) and no vaccine (placebo, line joining “x”) after beingintranasally challenged with 1×10⁶ PFU of the influenzaA/Swine/Iowa/15/30 (H1N1) virus.

FIG. 9 is a graph depicting virus titers in trachea/lung tissue of micereceiving VLP vaccine alone (line joining black diamonds); VLP and CpG(line joining black squares); inactivated virus vaccine (line joiningblack triangles) and no vaccine (placebo, line joining “x”) after beingintranasally challenged with 1×10⁶ PFU of the influenzaA/Swine/Iowa/15/30 (H1N1) virus. At days 2, 4, 6 and 8 post-challenge,four animals per group were sacrificed and trachea/lungs tissues wereharvested and virus titers determined by an MDCK cells based ELISA. Eachtime point represents the average titer of four mice and the verticallines indicate the standard deviations values.

FIG. 10 is a schematic depicting amino acid differences between swine(SEQ ID NOs:1, 3, 5 and 7) and 1918 (SEQ ID NOs:2, 4, 6 and 8) HAs.Antigenic sites ca, cb, sa and sb mapped on the HA1 globular portion ofthe H1subtype of HA are shown. See, Gerhard et al. (1981) Nature290(5808):713-717; Caton et al. (1982) Cell 31(2 Pt 1): 417-427.Numbered arrows (1 to 5) above residues indicate that five of the 22amino acid differences between the swine and 1918 virus on the HALmapped on the four antigenic sites. Numbered arrow “0” depicts a changeadjacent to the Sb site, which change may influence the antibodyinteractions with this site.

FIG. 11 is a schematic depicting the structure of exemplary sub-viralstructures (VLPs) as described herein. The matrix M1 protein underlinesthe membrane of the structure and chimeric glycoproteins HA (e.g., HA1,HA5 and/or HA7) and NA (e.g., NA1 and/or NA7) decorate the surface ofthe particle. The M2 protein spans the membrane and may carry adjuvantmolecule(s) linked to either the internal carboxyl or external aminoterminus.

FIG. 12 is a schematic depicting exemplary chimeric HA and NAglycoproteins in which the transmembrane and cytoplasmic domains of theselected HA and NA proteins are replaced with sequences from influenzavirus A/PR/8/34.

FIG. 13 depicts the nucleotide (SEQ ID NOs:9 and 10) and amino acidsequence (SEQ ID NO:11) of amino terminus of A/Udorn/72 (H3N2)Neuraminidase (NA) protein, including the cytoplasmic tail andtransmembrane domains. The NA cytoplasmic tail contains six N-terminalresidues (MNPNQK, shown in bold) (residues 1 to 6 of SEQ ID NO:11) whichare identical sequence almost all nine known NA subtypes. Thetransmembrane domain is underlined.

FIG. 14 depicts the nucleotide (SEQ ID NOs:12 and 13) and amino acidsequence (SEQ ID NO:14) of amino terminus of A/Udorn/72 hemagglutinin(HA) protein, including the cytoplasmic tail and transmembrane domains.The HA cytoplasmic tail contains 10-12 residues (QKGNIRCNICI, shown inbold) (residues 30 to 40 of SEQ ID NO:14) which are highly conservedbetween influenza strains. The transmembrane domain is underlined andthree residues of the ectodomain (YKD, residues 1-3 of SEQ ID NO:14) areshown in lower case.

FIG. 15 depicts the nucleotide (SEQ ID NO:15) and amino acid sequence(SEQ ID NO:16) of amino terminus of influenza A/PR/8/34 (H1N1)Neuraminidase (NA) protein, including the cytoplasmic tail andtransmembrane domains. The NA cytoplasmic tail contains six N-terminalresidues (MNPNQK, shown in bold) (residues 1 to 6 of SEQ ID NO:16) whichare identical sequence almost'all nine known NA subtypes. Thetransmembrane domain is underlined.

FIG. 16 depicts the nucleotide (SEQ ID NO:17) and amino acid (SEQ IDNO:18) sequence of amino terminus of AJPR/8/34 hemagglutinin (HA)protein, including the cytoplasmic tail and transmembrane domains. TheHA cytoplasmic tail (SNGSLQCRICI, residues 27 to 37 of SEQ ID NO:18) isshown in bold and the transmembrane domain is underlined.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning. A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularfoams “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a VLP” mayinclude a mixture of two or more such VLPs.

DEFINITIONS

As used herein, the terms “sub-viral particle” “virus-like particle” or“VLP” refer to a nonreplicating, viral shell, preferably derivedentirely or partially from influenza virus proteins. VLPs are generallycomposed of one or more viral proteins, such as, but not limited tothose proteins referred to as capsid, coat, shell, surface and/orenvelope proteins, or particle-forming polypeptides derived from theseproteins. VLPs can form spontaneously upon recombinant expression of theprotein in an appropriate expression system. Methods for producingparticular VLPs are known in the art and discussed more fully below. Thepresence of VLPs following recombinant expression of viral proteins canbe detected using conventional techniques known in the art, such as byelectron microscopy, biophysical characterization, and the like. See,e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J.Virol. (1994) 68:4503-4505. For example, VLPs can be isolated by densitygradient centrifugation and/or identified by characteristic densitybanding (e.g., Examples). Alternatively, cryoelectron microscopy can beperformed on vitrified aqueous samples of the VLP preparation inquestion, and images recorded under appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viral (e.g.,influenza) protein is meant a full-length or near full-length viralprotein, as well as a fragment thereof, or a viral protein with internaldeletions, insertions or substitutions, which has the ability to formVLPs under conditions that favor VLP formation. Accordingly, thepolypeptide may comprise the full-length sequence, fragments, truncatedand partial sequences, as well as analogs and precursor forms of thereference molecule. The term therefore intends deletions, additions andsubstitutions to the sequence, so long as the polypeptide retains theability to form a VLP. Thus, the term includes natural variations of thespecified polypeptide since variations in coat proteins often occurbetween viral isolates. The term also includes deletions, additions andsubstitutions that do not naturally occur in the reference protein, solong as the protein retains the ability to form a VLP. Preferredsubstitutions are those which are conservative in nature, i.e., thosesubstitutions that take place within a family of amino acids that arerelated in their side chains. Specifically, amino acids are generallydivided into four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serinethreonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune-system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the teen “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term includes polypeptides which includemodifications, such as deletions, additions and substitutions (generallyconservative in nature) as compared to a native sequence, so long as theprotein maintains the ability to elicit an immunological response, asdefined herein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present disclosure, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies by B-cells; and/or the activation of suppressorT-cells and/or γΔ T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic DNA sequences from viral or prokaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences, see e.g., McCaughan et al. (1995) PNAS USA92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.

A “nucleic acid” molecule can include, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting prokaryotic microorganisms or eukaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). Suitable programs for calculating the percent identity orsimilarity between sequences are generally known in the art.

A “vector” is capable of transferring gene sequences to target cells(e.g., bacterial plasmid vectors, viral vectors, non-viral vectors,particulate carriers, and liposomes). Typically, “vector construct,”“expression vector,” and “gene transfer vector,” mean any nucleic acidconstruct capable of directing the expression of one or more sequencesof interest in a host cell. Thus, the term includes cloning andexpression vehicles, as well as viral vectors. The term is usedinterchangeable with the terms “nucleic acid expression vector” and“expression cassette.”

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

General Overview

Described herein are virus-like particles (VLPs) comprising one or moreinfluenza proteins (e.g., glycoproteins, structural proteins, etc),compositions comprising these VLPs, as well as methods for making andusing these VLPs. The VLPs preferably comprise influenza matrix (M1and/or M2) proteins and influenza glycoproteins (hemagglutinin (HA)and/or neuraminidase (NA)). In certain embodiments, the VLPs comprisesurface glycoproteins (e.g., HA and/or NA) from the 1918 influenzastrain, the avian influenza virus A/Netherland/2003 (N7N7) and/or theavian influenza A virus H5N1. Methods of making and using thesecompositions are also described.

VLPs are structures that morphologically resemble an influenza virus,but are devoid of the genetic material required for viral replicationand infection. Using VLPs rather than inactivated influenza virus forthe production of VLP vaccines has several advantages, including ease ofproduction and purification, as compared current vaccines which aremanufactured in eggs Influenza VLP vaccine compositions may also avoidor reduce the unwanted side effects of current inactivated, egg-basedvaccines seen in young children, elderly, and people with allergies tocomponents of eggs. Furthermore, unlike many inactivated influenza virusvaccines, the HA and NA proteins of the VLPs described herein maintainconformational epitopes involved in eliciting protective neutralizingantibody responses.

When sequences encoding influenza proteins are expressed in eukaryotic,the proteins have been shown to self-assemble into noninfectiousvirus-like particles (VLP). See, Latham & Galarza (2001) J. Virol.75(13):6154-6165; Galarza et al. (2005) Viral. Immunol. 18(1):244-51;and U.S. Patent Publications 20050186621 and 20060263804. Thus, the useof VLP technology allows for the safe creation of vaccines againstextremely dangerous pathogens such as various human and avian influenzaviruses.

Virus-Like Particles

1. Influenza Polypeptide-Encoding Sequences

The VLPs produced as described herein are conveniently prepared usingstandard recombinant techniques. Polynucleotides encoding the influenzaproteins are introduced into a host cell and, when the influenzaproteins are expressed in the cell, they assemble into VLPs.

Polynucleotide sequences coding for molecules (structural and/or antigenpolypeptides) that form and/or incorporated into the VLPs can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. For example, plasmidswhich contain sequences that encode naturally occurring or alteredcellular products may be obtained from a depository such as theA.T.C.C., or from commercial sources. Plasmids containing the nucleotidesequences of interest can be digested with appropriate restrictionenzymes, and the released DNA fragments containing the nucleotidesequences can be inserted into a gene transfer vector using standardmolecular biology techniques.

Alternatively, cDNA sequences may be obtained from cells which expressor contain the sequences, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.Briefly, mRNA from a cell which expresses the gene of interest can bereverse transcribed with reverse transcriptase using oligo-dT or randomprimers. The single stranded cDNA may then be amplified by PCR (see U.S.Pat. Nos. 4,683,202, 4,683,195 and 4,800,159, see also PCR Technology:Principles and Applications for DNA Amplification, Erlich (ed.),Stockton Press, 1989)) using oligonucleotide primers complementary-tosequences on either side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

The influenza VLPs described herein are typically formed by expressingsequences encoding one or more influenza matrix proteins (M1 and,optionally, M2) and one or more antigenic influenza glycoproteins (HAand NA) in a host cell. The expressed proteins self-assemble into VLPswith the antigenic glycoproteins decorating the surface of the VLP.

The VLPs described herein may further comprise an influenzanucleoprotein (NP) and/or at least one protein of the polymerasecomplex, i.e., one or more of PB1, PB2 and PA (e.g., PB1, PB2, and PA;PB1 and PB2; PB1 and PA; PB2 and PA). Preferably, the VLPs do notinclude NP and all three proteins of the polymerase complex. Thestructure and function of influenza NP and polymerase complex proteinsis known and described for example on pages 428 to 432 of KubyImmunology, 4^(th) ed. (Goldsby et al. eds.) WH Freeman & Company, NewYork.

The sequences may encode naturally occurring or modified (e.g., bydeletions, additions and/or substitutions) influenza polypeptides andmay be obtained from any influenza virus strain. (see, Examples). Forexample, in certain embodiments, the sequences encoding the matrixprotein(s) are derived from the influenza virus strain A/Udorn/72 (H3N2or A/PR/8 (H1N1) and the glycoproteins are derived from the 1918influenza A virus strain (H1N1); avian influenza virusA/Mallard/Netherlands/2000 (H7N7); or avian influenza virusA/Vietnam/1203/2004 (H5N1).

In certain embodiments, the glycoprotein sequences encode chimericpolypeptides, for example influenza chimeric glycoproteins in which allor part (s) of the glycoproteins are replaced with sequences from otherviruses and/or sequences from other influenza strains. In a preferredembodiment, the chimeric glycoprotein-encoding sequences are modifiedsuch that the transmembrane and/or cytoplasmic tail domains are encodedby sequences derived from a different influenza strain than the strainfrom which the antigenic portion of the glycoprotein decorating thesurface of the VLP is derived. See, FIG. 12 and FIGS. 13 to 16. Forexample, the transmembrane domain and cytoplasmic tail of both HA and NAmay be replaced by the homologous domains derived from influenzaA/PR/8/34 (H1N1) or A/Udorn/72 (H3N2). The HA molecule is a type Iglycoprotein, thus the trans-membrane and cytoplasmic tail exchanged arelocated at the carboxyl-terminal (COOH₂) end of the molecule. In thecase of NA, a type II glycoprotein, the exchanged domains are located atthe amino-terminal (NH₂) end of the molecule (FIG. 12). These exchangesenhance the interaction of the surface glycoproteins with the scaffoldformed by the matrix protein M1, which is derived from either influenzaA/PR/8/34 or A/Udorn/72 virus and underlies the membrane of thesub-viral structure (FIGS. 11 to 16).

Preferably, the influenza sequences employed to form influenza VLPsexhibit between about 60% to 80% (or any value therebetween including61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78% and 79%) sequence identity to a naturally occurringinfluenza polynucleotide sequence and more preferably the sequencesexhibit between about 80% and 100% (or any value therebetween including81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% and 99%) sequence identity to a naturally occurringinfluenza polynucleotide sequence.

Any of the sequences described herein may further include additionalsequences. For example, to further to enhance vaccine potency, hybridmolecules are expressed and incorporated into the sub-viral structure.These hybrid molecules are generated by linking, at the DNA level, thesequences coding for the M1 or M2 genes with sequences coding for anadjuvant or immuno-regulatory moiety. During sub-viral structureformation, these chimeric proteins are incorporated into or onto theparticle depending on whether M1 or optionally included M2 carries theadjuvant molecule. The incorporation of one or more polypeptideimmunomodulatory polypeptides (e.g., adjuvants describe in detail below)into the sequences described herein into the VLP may enhance potency andtherefore reduces the amount of antigen required for stimulating aprotective immune response. Alternatively, as described below, one ormore additional molecules (polypeptide or small molecules) may beincluded in the VLP-containing compositions after production of the VLPfrom the sequences described herein.

These sub-viral structures do not contain infectious viral nucleic acidsand they are not infectious eliminating the need for chemicalinactivation. Absence of chemical treatment preserves native epitopesand protein conformations enhancing the immunogenic characteristics ofthe vaccine.

The sequences described herein can be operably linked to each other inany combination. For example, one or more sequences may be expressedfrom the same promoter and/or from different promoters. As describedbelow, sequences may be included on one or more vectors.

2. Expression Vectors

Once the constructs comprising the sequences encoding the influenzapolypeptides desired to be incorporated into the VLP have beensynthesized, they can be cloned into any suitable vector or replicon forexpression. Numerous cloning vectors are known to those of skill in theart, and one having ordinary skill in the art can readily selectappropriate vectors and control elements for any given host cell type inview of the teachings of the present specification and information knownin the art about expression. See, generally, Ausubel et al, supra orSambrook et al, supra.

Non-limiting examples of vectors that can be used to express sequencesthat assembly into VLPs as described herein include viral-based vectors(e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus),baculovirus vectors (see, Examples), plasmid vectors, non-viral vectors,mammalian vectors, mammalian artificial chromosomes (e.g., liposomes,particulate carriers, etc.) and combinations thereof.

The expression vector(s) typically contain(s) coding sequences andexpression control elements which allow expression of the coding regionsin a suitable host. The control elements generally include a promoter,enhancer, exon, intron, splicing sites translation initiation codon, andtranslation and transcription termination sequences, and an insertionsite for introducing the insert into the vector. Translational controlelements have been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M.,J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., MethodsEnzymol 60:360-375, 1979).

For example, typical promoters used for mammalian cell expressioninclude the SV40 early promoter, a CMV promoter such as the CMVimmediate early promoter (a CMV promoter can include intron A), RSV,HIV-LTR, the mouse mammary tumor virus LTR promoter (MMLV-LTR), FIV-LTR,the adenovirus major late promoter (Ad MLP), and the herpes simplexvirus promoter, among others. Other nonviral promoters, such as apromoter derived from the murine metallothionein gene, will also finduse for mammalian expression. Typically, transcription termination andpolyadenylation sequences will also be present, located 3′ to thetranslation stop codon. Preferably, a sequence for optimization ofinitiation of translation, located 5′ to the coding sequence, is alsopresent. Examples of transcription terminator/polyadenylation signalsinclude those derived from SV40, as described in Sambrook, et al.,supra, as well as a bovine growth hormone terminator sequence. Introns,containing splice donor and acceptor sites, may also be designed intothe constructs as described herein (Chapman et al., Nuc. Acids Res.(1991) 19:3979-3986).

Enhancer elements may also be used herein to increase expression levelsof the constructs, for example in mammalian host cells. Examples includethe SV40 early gene enhancer, as described in Dijkema et al., EMBO J.(1985) 4:761, the enhancer/promoter derived from the long terminalrepeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al.,Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived fromhuman CMV, as described in Boshart et al., Cell (1985) 41:521, such aselements included in the CMV intron A sequence (Chapman et al., Nuc.Acids Res. (1991) 19:3979-3986).

It will be apparent that a vector may contain one or more sequences asdescribed herein. For example, a single vector may carry sequencesencoding all the proteins found in the VLP. Alternatively, multiplevectors may be used (e.g., multiple constructs, each encoding a singlepolypeptide-encoding sequence or multiple constructs, each encoding oneor more polypeptide-encoding sequences). In embodiments in which asingle vector comprises multiple polypeptide-encoding sequences, thesequences may be operably linked to the same or differenttranscriptional control elements (e.g., promoters) within the samevector.

In addition, one or more sequences encoding non-influenza proteins maybe expressed and incorporated into the VLP, including, but not limitedto, sequences comprising and/or encoding immunomodulatory molecules(e.g., adjuvants described below), for example, immunomodulatingoligonucleotides (e.g., CpGs), cytokines, detoxified bacterial toxinsand the like.

3. VLP Production

As noted above, influenza proteins expressed in a eukaryotic host cellhave been shown to self-assemble into noninfectious virus-like particles(VLP). Accordingly, the sequences and/or vectors described herein arethen used to transform an appropriate host cell. The construct(s)encoding the proteins that form the VLPs described herein provideefficient means for the production of influenza VLPs using a variety ofdifferent cell types, including, but not limited to, insect, fungal(yeast) and mammalian cells.

Preferably, the sub-viral structure vaccines are produced in eukaryoticcells following transfection, establishment of continuous cell lines(using standard protocols as known to one skilled in the art) and/orinfection with DNA molecules that carry the influenza genes of interest.The level of expression of the proteins required for sub-viral structureformation is maximized by sequence optimization of the eukaryotic orviral promoters that drive transcription of the selected genes. Thesub-viral structure vaccine is released into the culture medium, fromwhere it is purified and subsequently formulated as vaccine. Thesub-viral structures are not infectious vaccines and therefore vaccineinactivation is not required.

The ability of influenza polypeptides expressed from sequences asdescribed herein to self-assemble into VLPs with antigenic glycoproteinspresented on the surface allows these VLPs to be produced in any hostcell by the co-introduction of the desired sequences. The sequence(s)(e.g., in one or more expression vectors) may be stably and/ortransiently integrated in various combinations into a host cell.

Suitable host cells include, but are not limited to, bacterial,mammalian, baculovirus/insect, yeast, plant and Xenopus cells.

For example, a number of mammalian cell lines are known in the art andinclude primary cells as well as immortalized cell lines available fromthe American Type Culture Collection (A.T.C.C.), such as, but notlimited to, BHK, VERO, HT1080, MRC-5, WI 38, MDCK, MDBK, 293, 293T, RD,COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1,CEM, myeloma cells (e.g., SB20 cells) and CEMX174 (such cell lines areavailable, for example, from the A.T.C.C.).

Similarly, bacterial hosts such as E. coli, Bacillus subtilis, andStreptococcus spp., will find use with the present expressionconstructs.

Yeast hosts useful in the present disclosure include inter alia,Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactic, Pichiaguillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowialipolytica. Fungal hosts include, for example, Aspergillus.

Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni. See, Latham &Galarza (2001) J. Virol. 75(13):6154-6165; Galarza et al. (2005) Viral.Immunol. 18(1):244-51; and U.S. Patent Publications 20050186621 and20060263804.

Cell lines expressing one or more of the sequences described above canreadily be generated given the disclosure provided herein by stablyintegrating one or more sequences (expression vectors) encoding theinfluenza proteins of the VLP. See, Example 7. The promoter regulatingexpression of the stably integrated influenza sequences (s) may beconstitutive or inducible. Thus, a cell line can be generated in whichone or more both of the matrix proteins are stably integrated such that,upon introduction of the influenza glycoprotein-encoding sequencesdescribed herein (e.g., chimeric glycoproteins) into a host cell andexpression of the influenza proteins encoded by the polynucleotides,non-replicating influenza viral particles that present antigenicglycoproteins are formed.

Example 7 describes the production of a mammalian (CHO) cell line thatwas generated to express an influenza HA glycoprotein. Sequencesencoding a matrix protein (M1 and/or M2) and/or NA can be introducedinto such a cell line to produce VLPs as described herein.Alternatively, a cell line that stably produces influenza M1 and,optionally, M2 proteins can be generated and sequences encoding theglycoprotein(s) from the selected influenza strain introduced into thecell line, resulting in production of VLPs presenting the desiredantigenic glycoproteins.

The parent cell line from which an influenza VLP-producer cell line isderived can be selected from any cell described above, including forexample, mammalian, insect, yeast, bacterial cell lines. In a preferredembodiment, the cell line is a mammalian cell line (e.g., 293, RD,COS-7, CHO, BHK, VERO, MRC-5, HT1080, and myeloma cells). Production ofinfluenza VLPs using mammalian cells provides (i) VLP formation; (ii)correct myristylation, glycosylation and budding; (iii) absence ofnon-mammalian cell contaminants and (iv) ease of purification.

In addition to creating cell lines, influenza-encoding sequences mayalso be transiently expressed in host cells. Suitable recombinantexpression host cell systems include, but are not limited to, bacterial,mammalian, baculovirus/insect, vaccinia, Semliki Forest virus (SFV),Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis (VEE)),mammalian, yeast and Xenopus expression systems, well known in the art.Particularly preferred expression systems are mammalian cell lines,vaccinia, Sindbis, insect and yeast systems.

Many suitable expression systems are commercially available, including,for example, the following: baculovirus expression (Reilly, P. R., etal., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames,et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto,Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expressionof proteins in mammalian cells using vaccinia” In Current Protocols inMolecular Biology (F. M. Ausubel, et al. Eds.), Greene PublishingAssociates & Wiley Interscience, New York (1991); Moss, B., et al., U.S.Pat. No. 5,135,855, issued Aug. 4, 1992), expression in bacteria(Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons, Inc., Media Pa.; Clontech), expression in yeast(Rosenberg, S, and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued,Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S.Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93(1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D.V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R Fink,Methods in Enzymology 194 (1991)), expression in mammalian cells(Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)), and expression in plant cells (plantcloning vectors, Clontech Laboratories, Inc., Palo-Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997).

When expression vectors containing the altered genes that code for theproteins required for sub-viral structure vaccine formation areintroduced into host cell(s) and subsequently expressed at the necessarylevel, the sub-viral structure vaccine assembles and is then releasedfrom the cell surface into the culture media (FIG. 11).

Depending on the expression system and host selected, the VLPs areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptides are expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art. If the VLPs are accumulateintracellularly, the cells are then disrupted, using chemical, physicalor mechanical means, which lyse the cells yet keep the VLPssubstantially intact. Such methods are known to those of skill in theart and are described in, e.g., Protein Purification Applications: APractical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).Alternatively, VLPs may be secreted and harvested from the surroundingculture media.

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by densitygradient centrifugation, e.g., sucrose gradients, PEG-precipitation,pelleting, and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

Compositions

VLPs produced as described herein can be used to elicit an immuneresponse when administered to a subject. As discussed above, the VLPscan comprise a variety of antigens (e.g., one or more influenza antigensfrom one or more strains or isolates). Purified VLPs can be administeredto a vertebrate subject, usually in the form of vaccine compositions.Combination vaccines may also be used, where such vaccines contain, forexample, other subunit proteins derived from influenza or otherorganisms and/or gene delivery vaccines encoding such antigens.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 10 (or more) mg, morepreferably about 1 μg to about 300 μg, of VLP/antigen.

Sub-viral structure vaccines are purified from the cell culture mediumand formulated with the appropriate buffers and additives, such as a)preservatives or antibiotics; b) stabilizers, including proteins ororganic compounds; c) adjuvants or immuno-modulators for enhancingpotency and modulating immune responses (humoral and cellular) to thevaccine; or d) molecules that enhance presentation of vaccine antigensto specifics cell of the immune system. This vaccine can be prepared ina freeze-dried (lyophilized) form in order to provide for appropriatestorage and maximize the shelf-life of the preparation. This will allowfor stock piling of vaccine for prolonged periods of time maintainingimmunogenicity, potency and efficacy.

A carrier is optionally present in the compositions described herein.Typically, a carrier is a molecule that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycollic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J.Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine11(2):149-54, 1993. Such carriers are well known to those of ordinaryskill in the art.

Additionally, these carriers may function as immunostimulating agents(“adjuvants”). Exemplary adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu); (3)saponin adjuvants, such as Stimulon™. (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S 109(where serine is substituted for the wild-type amino acid at position109), and PT-K9/G129 (where lysine is substituted for the wild-typeamino acid at position 9 and glycine substituted at position 129) (see,e.g., International Publication Nos. WO93/13202 and WO92/19265); and (7)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Examples of suitable immunomodulatory molecules for use herein includeadjuvants described above and the following: IL-1 and IL-2 (Karupiah etal. (1990) J. Immunology 144:290-298, Weber et al. (1987) J. Exp. Med.166:1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224, andU.S. Pat. No. 4,738,927-); IL-3 and IL-4 (Tepper et al. (1989) Cell57:503-512, Golumbek et al. (1991) Science 254:713-716, and U.S. Pat.No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J. Immunol.139:4116-4121, and International Publication No. WO 90/06370); IL-7(U.S. Pat. No. 4,965,195); IL-8, IL-9, IL-10, IL-11, IL-12, and IL-13(Cytokine Bulletin, Summer 1994); IL-14 and IL-15; alpha interferon(Finter et al. (1991) Drugs 42:749-765, U.S. Pat. Nos. 4,892,743 and4,966,843, International Publication No. WO 85/02862, Nagata et al.(1980) Nature 284:316-320, Familletti et al. (1981) Methods in Enz.78:387-394, Twu et al. (1989) Proc. Natl. Acad. Sci. USA 86:2046-2050,and Faktor et al. (1990) Oncogene 5:867-872); β-interferon (Seif et al.(1991) J. Virol. 65:664-671); γ-interferons (Watanabe et al. (1989)Proc. Natl. Acad. Sci. USA 86:9456-9460, Gansbacher et al. (1990) CancerResearch 50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother.30:34-42, and U.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat.Nos. 4,999,291 and 4,810,643); GM-CSF (International Publication No. WO85/04188); tumor necrosis factors (TNFs) (Jayaraman et al. (1990) J.Immunology 144:942-951); CD3 (Krissanen et al. (1987) Immunogenetics26:258-266); ICAM-1 (Altman et al. (1989) Nature 338:512-514, Simmons etal. (1988) Nature 331:624-627); ICAM-2, LFA-1, LFA-3 (Wallner et al.(1987) J. Exp. Med. 166:923-932); MHC class 1 molecules, MHC class IImolecules, B7.1-β2-microglobulin (Parnes et al. (1981) Proc. Natl. Acad.Sci. USA 78:2253-2257); chaperones such as calnexin; and MHC-linkedtransporter proteins or analogs thereof (Powis et al. (1991) Nature354:528-531). Immunomodulatory factors may also be agonists,antagonists, or ligands for these molecules. For example, soluble formsof receptors can often behave as antagonists for these types of factors,as can mutated forms of the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids encoding one or more of the above-identified polypeptides canbe digested with appropriate restriction enzymes, and DNA fragmentscontaining the particular gene of interest can be inserted into a genetransfer vector (e.g., expression vector as described above) usingstandard molecular biology techniques. (See, e.g., Sambrook et al.,supra, or Ausubel et al. (eds) Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience).

Administration

The VLPs and compositions comprising these VLPs can be administered to asubject by any mode of delivery, including, for example, by parenteralinjection (e.g. subcutaneously, intraperitoneally, intravenously,intramuscularly, or to the interstitial space of a tissue), or byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g.see WO99/27961) or transcutaneous (e.g. see WO02/074244 andWO02/064162), intranasal (e.g. see WO03/028760), ocular, aural,pulmonary or other mucosal administration. Multiple doses can beadministered by the same or different routes. In a preferred embodiment,the doses are intranasally administered.

The VLPs (and VLP-containing compositions) can be administered prior to,concurrent with, or subsequent to delivery of other vaccines. Also, thesite of VLP administration may be the same or different as other vaccinecompositions that are being administered.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be deteimined by thepotency of the modality, the vaccine delivery employed, the need of thesubject and be dependent on the judgment of the practitioner.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting. For instance,although the VLPs disclosed in the Examples include M2, it will beapparent from the above disclosure that M2 is optional and thatinfluenza VLPs as described herein can be formed without M2. See, also,U.S. Patent Publication Nos. 20050186621 and 20060263804.

EXAMPLES Example 1 Generation of a Baculovirus Transfer Vector

The HA gene of the influenza virus A/South Carolina/1/1918 (H1N1)(GenBank Accession #AF117241) was de novo synthesized with the followingmodifications: 1) the 3′ terminus which encodes the transmembrane domainand cytoplasmic tail were replaced with the analogous domains of the HA3of influenza A/Udorn/72 (H3N1), 2) the Not I, Kpn I restriction sites aswell as the T7 polymerase promoter sequence were added at the 5′terminus of gene whereas Avr II and Not I were added to the 3′ terminus.

The nucleotide sequence of the A/Brevig_Mission/1/1918 (H1N1)neuraminidase (NA) gene (GenBank Accession #AF250356) was also de novosynthesized with the following changes: 1) the 5′ terminus encoding theNH2-terminal cytoplasmic tail and transmembrane anchoring domain werereplaced with those of the NA1 of influenza A/Udorn/72 (H3N1), 2) theSma I and Asc I restriction sites were added to the 5′ end of the genewhereas the FseI, Sma I and Not I were added at the 5′ terminus. The NAgene was sub-cloned via the Sma I site into the intermediate shuttlevector as described in Latham & Galarza (2001) J. Virol.75(13):6154-6165 and U.S. Patent Publications 20050186621, which carriesthe M1 gene. In this intermediate construct, the NA gene is positionedunder the transcriptional control of the baculovirus p10 promoter andthe M1 gene under the transcriptional control of the polyhydronpromoter.

These M1 and NA1 genes and respective promoters were cut out as a singlefragment by digesting the shuttle vector with PmeI and SacI restrictionenzymes. The M1-NA insert was then sub-cloned into the PmeI/SacI sitesof the baculovirus transfer vector pAcab4-M2 (Latham & Galarza (2001) J.Virol. 75(13):6154-6165 and U.S. Patent Publication 20050186621), whichcontains the M2 gene was already cloned.

Subsequently, the 1918 HA (HA1N1) gene was sub-cloned into thepAcAB4-M2-M1-NA via the Not I site. Restriction enzyme analysis was usedto select the plasmids that carried the HA gene in the correctorientation with respect the polyhedron promoter. The selectedpAcAB4-M2-1918HA-1918NA-M1 plasmid was amplified, purified using anendotoxin-free plasmid purification kit from Qiagen, Valencia, Calif.and sequenced to verify the genes and promoters nucleotide sequence aswell as the genes orientation.

In this final construct, the M1 and 1918HA genes are under the controlof the polyhedrin promoter and in opposite direction whereas the M2 and1918NA are also in opposite direction to each other and under thecontrol of the p10 promoter (FIG. 1).

Example 2 Generation of a Baculovirus Recombinant Virus that Producesthe 1918 Virus-Like Particles (VLP)

To create a baculovirus recombinant, Sf9 insect cells were seeded at adensity of ˜2×10⁶ onto 60 mm dishes and subsequently transfected with amixture of ˜2 μg of the 1918-transfer vector and 0.5 μg of linearizedBaculoGold Bright™ baculovrius DNA, BD Biosciences Pharmagin (San Diego,Calif.). This linear baculovirus DNA carries within its genome the greenfluorescence protein (GFP) gene, therefore the infectious virusgenerated by homologous recombination within the insect cells expressesthe GFP in addition to the influenza proteins required for VLP assembly.The GFP protein serves as a marker for recognizing and selecting virusrecombinants as well as for accurate determination of virus titers andmultiplicity of infections.

GFP producing baculovirus recombinants were selected, expanded, andanalyzed by PCR and Western blot to verify the presence of the fourgenes and their expression respectively. The selected recombinant viruswas amplified and subsequently titrated in Sf9 cells by using as readoutthe microscopic detection of GFP in the highest of triplicate endpointdilutions.

Example 3 Production and Purification of the 1918 VLP Vaccine

Sf9 cells were grown in shaker flasks with serum-free medium at 28° C.For vaccine production, cells were infected with the recombinantbaculovirus at a multiplicity of infection (MOI) of 1, in 1/10 of thefinal culture volume. Virus absorption to cells was allowed for 1 hourat which point fresh medium was added to bring the culture to its finalvolume. Progression of the infection was monitored by taking cellsamples and observing, under the microscope, the expression of the GFPby fluorescence microscopic examination of infected cells was performedat 20, 40 and 60 h post infection to determine the percentage of cellsexpressing GFP and the intensity of the GFP signal. Because the GFP geneis carried within the genome of the recombinant virus that expresses theVLP forming proteins, its expression denotes production of the 1918 VLPvaccine by the infected cells (FIG. 2).

After 96 hours post infection, the culture supernatant, which containsthe vaccine, was separated from the cells by low speed centrifugation(2000×g for 15 min at 4° C.). Then, the vaccine particles were pelletedby centrifugation of the supernatant at 200,000×g for 90 min Dependingon the number of cells initially infected, the vaccine pellet wasresuspended in 0.5 or 1 ml of 1× phosphate buffered saline (PBS),homogenized by a brief sonication and then loaded on top of an iodixanol(Optiprep, Nycomed) gradient (density of 1.08 to 1.32 g/ml). Thegradient was spun at 200,000×g for 3 h and the vaccine particles underthese conditions form a band within the top ⅓ of the gradient from wherethey were collected. See, Latham & Galarza (2001) J. Virol.75(13):6154-6165; Galarza et al. (2005) Viral. Immunol. 18(1):244-51;and U.S. Patent Publication 20050186621. Vaccine particles were dialyzedin 1×PBS and this preparation was used as VLP vaccine with and withoutadjuvant.

Example 4 Western Blot Analysis of Purified VLP Vaccine

Purified vaccine material was analyzed by Western blot to confirm thepresence of specific proteins and determine the HA content as previouslyperformed essentially as described in Galarza et al. (2005) Viral.Immunol. 18(1):244-51. Briefly, the protein content of the 1918 VLPvaccine was evaluated with antibodies to the 1918 HA and M1 proteins. Amouse monoclonal anti-1918 HA antibody was used as primary antibody todetect the 1918 HA. Rabbit anti-mouse horseradish peroxidase conjugated(BioRad, Hercules, Calif.) was used as secondary antibody. Similarly, amouse polyclonol anti-M1 antibody (AbD Serotec, Raleigh, N.C.) was usedto detect the M1 protein, which was derived from the influenzaA/Udorn/72. The M1 and M2 proteins share nine amino acids at their NH2terminals and a band of the size expected for the M2 protein wasobserved with very high levels of VLP and M1 antibody.

Western blot analyses demonstrated that the 1918 HA protein was presentin the purified 1918 VLP vaccine fractions (FIG. 3, panel A). When anantibody to the M1 protein was used in Western blot, it was demonstratedthat M1 protein was present in the same two VLP vaccine fractions as theHA (FIG. 3, panel B). The M2 protein was detected when higherconcentrations of purified material were loaded in the gel (Data notshown). Expression and incorporation of the NA protein was evaluated inpurified VLPs by a neuraminidase assay (FIG. 5), which showed that NAwas indeed incorporated into the VLP as previously shown byimmunofluorescence and immunogold labeled electron microscopy withanother VLP (Latham & Galarza (2001) J. Virol. 75(13):6154-6165).

Example 5 Neuraminidase Assay

The presence of neuraminidase (NA) was evaluated by detecting itsenzymatic activity (sialidase) which cleaves the terminal sialic acidresidues from glycoproteins essentially as described in the WHO manualon animal influenza diagnosis and surveillance. Global InfluenzaProgramme. Geneva, World Health Organization. 2002: p. 40-47. PurifiedVLPs were incubated with fetuin as substrate for 16 hours at 37° C. Theamount of free sialic acid released from the substrate by the enzymaticactivity of the NA was detected with thiobarbituric acid which producesa pink color in proportion to the amount of free sialic acid in theassay. A reaction with PBS and fetuin was carried out as control andused as blank in the spectrophotometric readings. Influenza A/swinevirus was tested as positive control. Color intensity was measuredspectrophotometrically at the wavelength of 549 nm. NA enzymaticactivity is expressed as optical density (OD) at a 549 nm

Example 6 VLP Vaccine Hemagglutination Assay

The ability of the 1918 VLPs vaccine to agglutinate red blood cells(RBC) was evaluated in a standard hemagglutination assay using cellsfrom two different species, chicken and turkey. Briefly, twofold serialdilutions of the purified VLP vaccine were carried out with 1×PBS inV-shape 96 well plates. Then, an equal volume of a 0.5% solution of RBCsin 1×PBS was added to the wells and the plate incubated at 4° C. for 1h. After this time, the appearance of a RBCs precipitate (RBCs button)indicates lack of hemagglutination. Hemagglutination titers areexpressed as the inverse of the highest dilution of the vaccine able toagglutinate RBCs.

Both chicken and turkey RBCs express on their surface a mixture of HAreceptor with sialic acid linked to galactose by either an α-2, 3(SAα-2,3) or an α-2, 6 linkage (SAα-2, 6). See, Iwatsuki-Horimoto et al.(2004) J Gen Virol 85(Pt 4):1001-1005; Stephenson et al. (2004) VirusRes. 103(1-2):91-95. The 1918 VLPs were functional in hemagglutinationwith turkey RBCs but were not functional with chicken RBCs, even at thelower dilutions (FIG. 4).

Example 7 Generation of CHO Cells that Express the 1918 HA Proteins

As an alternative source of antigen, a CHO cell line that expresses the1918 HA protein, was created by cloning the HA gene (see Example 1) intothe plasmid pcDNA 3.1/v5-His (Invitrogen, Chicago, Ill.) by ligationinto the XbaI/KpnI restriction sites, which were added to the ends ofthe synthesized HA genes by PCR. Purified recombinant plasmid DNA wastransfected into CHO cells using Lipofectamine 2000 reagent(Invitrogen). Subsequently, transfected cells were cultured in serumfree medium containing neomycin at the concentration of 2 mg/ml asselecting agent (a kill curve with normal CHO showed that 1 mg/ml ofneomycin kill all the cells in 7 days). Cells were subcultured underselective pressure conditions for 28 days and then tested for HA 1918expression.

Example 8 In Vivo Vaccination with Influenza VLPs

The immunogenicity and protective efficacy of the 1918 VLP vaccine wastested in 7-8 week old, female BALB/C mice (Charles River Laboratories,Wilmington, Mass.). Mice were housed in the Department of ComparativeMedicine, New York Medical College and the study was carried outfollowing institutional IACUC-approved protocols.

Four groups of 12 mice each received vaccine, placebo, and controltreatment. The two vaccine groups received (via intranasal route) twodoses, two weeks apart, of VLP vaccine (1 pg of HA content per dose)alone or admixed with 10 μg per dose of a oligonucleotide (20mer ODN).This ODN contained two CpG motifs located in the middle of the moleculeand spaced by two bases (CGXXCG) and flanked by complementary sequencesable to form a stem-loop structure. The placebo and control groups alsoreceived via intranasal route two doses of either PBS) or formalininactivated influenza A/Swine/Iowa/15/30 (H1N1) (˜1 μg of HA content),respectively.

Two weeks after the primary and booster immunizations, mice wereanesthetized with Ketamine-Xylazine (70 mg/kg and 6 mg/kg b.w.respectively) and blood samples were collected via retro-orbitalbleeding. Eighteen days after the booster immunization, mice in allgroups (anesthetized as above) were challenged via the intranasal routewith 1×10⁶ PFU per 20 μl of the influenza virus A/Swine/Iowa/15/30(H1N1), a surrogate for the extinct 1918 influenza virus. The influenzaASwine/Iowa/15/30 (H1N1) virus (VR-333, from ATCC, Manassas, Va.) isantigenically related and most contemporary to the 1918 influenza virusand was used as surrogate challenge. This virus was grown in 10 day-oldembryonated chicken eggs (SPF, Charles River Laboratories, MA) for 48hspost infection at 37° C. The infected allantoic fluid was harvested andclarified by low speed centrifugation and virus titer determined by theplaque assay in MDCK Cells. Aliquots of the virus were stored at −80° C.until use.

The challenge dose was delivered by small drops (10 μl per nostril)using a pipetman with an ultra slim capillary sequencing tip. Mice weremonitored daily for weigh loss, clinical signs and severity ofinfection. On days 2, 4, 6 and 8 post-challenge, four mice from eachgroup were euthanized and their nasal passages and trachea-lungs wereharvested and placed in 1.5 ml of SPG (0.22M sucrose, 0.01M potassiumphosphate, 0.005M potassium glutamate in phosphate buffered saline, pH7.2). Tissues were homogenized for 1.5 min with an Omni International THhomogenizer equipped with a saw-tooth generator. Samples werecentrifuged at 2500×g for 10 min to pellet cell debris and clarifiedsupernatant stored at −80° C. until virus titrations were performed.

Virus load in the nasal passages and trachea/lung tissues weredetermined by a cell-based ELISA assay using an anti-NP antibody thatrecognizes the nucleoprotein (NP) of influenza A viruses. This assay isvery sensitive and highly specific allowing for the detection of lowlevels of NP protein expressed in infected cells. MDCK cells were seededat the concentration of 5×10⁴ cells per well in 96 well tissue cultureplates and incubated overnight at 37° C. in 5% CO₂. Cell monolayers werewashed with PBS and infected with 100 μl of ten-fold serial dilutions inPBS of tissue homogenates. Lung/trachea and nasal tissue samples wereassayed in septaplicates on the same 96 well plate. Infected plates wereincubated at RT for 1 h and then the inoculum was removed and replacedwith 100 μl of MEM (Minimal Essential Medium) containing 50 U/mlpenicillin and 50 mg/ml streptomycin and 1 μg/ml trypsin TPCK(Worthington Biochemical, Lakewood N.J.). Subsequently, plates wereincubated at 37° C. in 5% CO₂ for an additional 40 hours, at which timethe plates were spun down at 2800×g for 12 min and then fixed for 10 minat RT with 100 μl of an acetone/methanol (1:1) mixture.

Plates were then washed 6 times with buffer (PBS with 0.05% tween-20),blocked for 1 h at RT with 1500 of blocking solution [5% nonfat milk, 1%bovine serum albumin (BSA) (Pierce, Rockford Ill.), 2% normal goat serum(Vector Labs, Burlingame Calif.), 0.05% tween-20 in PBS] and washedagain 3 times with wash buffer. Subsequently, 100 μl of a mousemonoclonal anti-NP primary antibody (Influenza A H1N1 clone IVF8Biodesign International, Saco, Me.; diluted 1:3000 in 1% BSA, 2% normalgoat serum, 0.05% tween-20 in PBS) was added and then plates incubatedfor 1 h at RT. Primary antibody was not added to rows 6 and 12 so thatbackground signals can be subtracted during virus titer calculation.Plates were again washed 3 times with wash buffer and then 1000 of asecondary goat anti-mouse antibody conjugated with HRP (1:2500 dilutionin 1% BSA, 2% normal goat serum, 0.05% tween-20 in PBS) (Bio-Rad,Hercules Calif.) was added and incubated for 1 h at RT. Again, plateswere washed 6 times with wash buffer and 100 μl of Ultra-TMB (Pierce,Rockford Ill.) was added and rocked at RT until color development. Thereaction was stopped by adding 100 μl of a 0.2M HCl acid solution whendesired color intensity was achieved. Absorbance in each plate wasmeasured at 450 nm with a Thermo Multiskan EX plate reader. Virus titerswere expressed as TCID₅₀ and were calculated according to theReed-Muench method (Reed (1938) Am. J. Hyg. 27:493-497.

A. Humoral Immune Response

The antibody response elicited by VLP vaccination was evaluated by ELISAusing 96 well plates (Immulon II, Thermo Lab Systems, Franklin, Mass.)coated with either detergent disrupted purified influenza virusA/Swine/Iowa/15/30 (H1N1) or CHO cells expressing the 1918 HA protein asantigens (Example 7). Virus coated plates received 50 ng of total viralprotein per well whereas the CHO cell based ELISA plates were seededwith an equivalent number of cells and incubated until they reachedconfluency at which time they were fixed with a 1:1 mixture ofmethanol/acetone for 10 min at RT. Both ELISA plates were blocked for 1h at RT with PBS solution, pH 7.2, containing 1% bovine serum albumin(BSA), 2% goat serum, 2% nonfat milk and 0.05% Tween-20 and subsequentlywashed 3 times with PBS containing 0.05% Tween-20 (PBST). Serialdilutions of individual serum samples were applied to either of theplates and incubated for 1 h at RT, followed by three washes with thePBST solution.

Subsequently, the plates were incubated for 1 h at RT with 100 μl of ahorseradish peroxidase (HRP) goat anti-mouse secondary antibody, diluted1:1000 in PBS plus 1% BSA, 2% goat serum and 0.05% Tween-20 and this wasfollowed by another set of three washes with PBST. Finally, the plateswere incubated with 100 μl of TMB solution (Pierce, Rockford, Ill.) andmonitored for color development. The color reaction was stopped byadding 1000 of 0.1M HCL. The absorbance was determined at 450 nm using aThermo Multiskan EX plate reader.

Absorbance titers were determined as the highest serum dilution that hadan optical density twice the absorbance given by the pre-immunizedcontrol serum.

Furthermore, the 1918 HA-CHO cell based ELISA was validated by utilizingCHO cells that expressed the 1918 HA, parental CHO cells that did notexpressed HA, an anti-1918 HA mouse monoclonal antibody as positivecontrol serum (see above) and mouse pre-immunization serum as negativecontrol. Using the two cell lines and antibodies, a standard curve wasestablished which verified the validity of the assay.

As shown in FIG. 6, the 1918 VLP vaccine groups produced high levels ofantibodies. When the CHO cells expressing the 1918 HA were used asantigen in the ELISA test, as also found with the disrupted virus ELISA,there was not a significant difference in the levels of serum IgGbetween mice immunized with the 1918 VLP vaccine alone or adjuvantedwith the CpG-ODN.

In addition, mice vaccinated with the 1918 VLP vaccine with or withoutCpG as well as mice immunized with the inactivated virus demonstrated anantibody response when disrupted virus was used as antigen (FIG. 6). Asshown in FIG. 6, CpG did not enhance the serum antibody response to the1918 VLP vaccine. The more robust antibody response in mice immunizedwith inactivated virus may be due to antigenic differences between the1918 HA, which is part of the VLP vaccine, and the HA of the swine viruswhich is the coating antigen for the virus ELISA (FIG. 10) and/or thatthe inactivated virus vaccine delivers a larger number of viral specificproteins/antigens (e.g. nucleoprotein NP) than the number of antigenspresent in the 1918 VLP vaccine that enhance the overall immune responsein the ELISA assay.

B. Challenge with Swine Influenza

To evaluate the level of protection afforded by two doses of the 1918VLP vaccine, formulated in PBS alone or in PBS plus CpG-ODN, inactivatedvirus control, and placebo inoculations, administered via intranasalroute, mice in all groups were challenged seventeen days after thesecond immunization with 1×10⁶ PFU of influenza A/Swine/Iowa/15/30(H1N1) virus. This influenza virus was selected as a surrogate challengebecause it is the most contemporary and antigenically related to the1918 virus currently available, other than the recently reconstructed1918 virus (Glaser et al. (2005) J. Virol. 79(17):11533-11536).

Preliminary experiments in mice showed that the swine virus at the doseutilized as challenge causes severe influenza illness with typicalclinical signs of the disease such as progressive inactivity beginningaround day 3 post infection, ruffled fur, labored breathing, tendency tohuddle and reduced or absence of water and food intake leading to severeweight loss. All these parameters were subsequently monitored during theprotective efficacy study with the 1918 VLP vaccine.

Vaccinated and control mice were slightly anesthetized, as describedabove, prior to receiving the virus challenge (1×10⁶ PFU), which wascontained in a volume of 20 μl and administered by intranasalinstillation of very small droplets delivered by ultra-slim sequencetips (10 μl per nostril). Following virus challenge, three differentmeasurements were utilized to assess vaccine protective efficacy: bodyweight measurement, clinical sign of illness and virus titers in nasaland trachea/lungs tissues. Clinical signs of influenza illness werescored as: (+) no clinical sign of disease, although body weightmeasurement may indicate slight body mass losses; (++) ruffled fur,inactivity, tendency to huddle; (+++) hunched back, pronounced ruffledfur, severed inactivity, (++++) labored breathing (high frequency andabdominal panting) severe hunched back, ruffled fur, complete inactivity(no response to stimulation) and severed weight loss.

Group average of daily weight measurements showed that mice immunizedwith 1918 VLP vaccine plus CpG experienced a slight decrease in bodyweight between days 4 and 8 post challenge, without clinical sign ofinfluenza infection, score (+) (FIG. 7); however the weight did not droplower than their starting weight at day 0. The mice in the 1918 VLPvaccine alone experienced a more pronounced reduction of body weightthat continued until day 6 at which point the trend reversed and micebegan to gain weight. Mice in this group, however, showed very minorclinical signs of illness (score ++), quite opposite to the inactivatedvirus control group which showed more severe clinical sign of disease(score between ++ and +++) with a less pronounced body weight losstendency until day 6 when these mice also began gaining weight (FIG. 7).The placebo control group showed a pronounced weight loss whichpersisted beyond day 6 and stabilized around day 8 without a clearrecovery at day 15 when the experiment was terminated. Symptoms werequite severe in this group (score++++) and one mouse died at day fivepost challenge. At 2, 4, 6 and 8 days post challenge, four animals pergroup were sacrificed and virus titers in the nasal tissue as well as inthe trachea/lungs were determined in a cell based assay. Hence, thescore of clinical signs and group average of body weight reported aftereach time point were those collected from the remaining animals in eachgroup.

Mice immunized with the 1918 VLP vaccine alone or with the 1918 VLP plusCpG had significantly lower virus titers in the nasal tissue at days 2(P<0.0005), 4 (P<0.05) and 6 (P<0.005) post challenge as compared to theplacebo group (FIG. 8). At day 6 post challenge, the 1918 VLP plus CpGvaccinated mice showed complete clearance of the swine virus whereas theVLP alone vaccine group still showed virus in the nasal tissue (FIG. 8).Mice immunized with the inactivated virus also had a significant lowertiter than the placebo control at days 2 (P<0.005) and 4 (P<0.05) postchallenge; however these titers were slightly higher on average than thevirus load detected in the nose of mice immunized with either of the two1918 VLP vaccines (FIG. 8). Virus was not detected in the nose of any ofthe groups at day 8 post challenge.

The level of protection of the lower respiratory track provided byeither of the two 1918 VLP vaccine formulations, the inactivated virusor the placebo treatment, was also evaluated by assessing virus loads inthe trachea/lungs tissues at days 2, 4, 6 and 8 post challenge. Thisshowed that mice vaccinated with the 1918 VLP vaccine alone or 1918 VLPplus CpG, had significantly lower virus titers in the trachea/lungtissues, at days 2 (P<0.05), 4 (P<0.05) and 6 (P<0.005) post challengethan mice in the placebo group (FIG. 9). Furthermore, mice immunizedwith the 1918 VLP vaccine plus CpG were able to completely clear theviral infection from the lower respiratory track by day 6 postchallenge, whereas mice in the 1918 VLP vaccine alone and inactivatedvirus groups still had virus at this time, although at significantlylower titers than the placebo control group. Virus titers in thetrachea/lungs of mice immunized with the 1918 VLP vaccine alone were atday 4 significantly lower than that of mice immunized with theinactivated virus control, however at days 2 and 6 they showed similarlevels of virus which was cleared by day 8 in both groups as well as inthe placebo control.

Even though virus was detected in mice vaccinated either with the 1918VLP alone, 1918 VLP plus CpG or inactivated swine virus vaccine, at day2 post challenge there was a significant difference in virus titers byabout a 1 or 2 log difference in the trachea/lung and nasal tissues,respectively, as compared to the placebo group. The data also indicatedthat the viral load was higher in the lower respiratory tract than inthe upper tract but complete virus clearance occurred at day 8 postchallenge in both upper and lower tracts of all the groups.

Thus, VLP vaccination conferred a significant protection againstchallenge with the swine virus.

What is claimed is:
 1. A VLP comprising influenza proteins, theinfluenza proteins consisting essentially of an influenza M1 protein, aninfluenza M2 protein, a first influenza HA protein and a first influenzaNA protein.
 2. The VLP of claim 1, wherein the HA and NA proteinscomprise HA1 and NA1.
 3. The VLP of claim 1, wherein the HA and NAproteins comprise HA7 and NA7.
 4. The VLP of claim 1, wherein the HA andNA proteins comprise HA5 and NA1.
 5. The VLP of claim 1, wherein: (i) aportion of the first influenza HA protein is replaced with a homologousregion from second influenza HA protein, wherein the second influenza HAprotein is from a different strain than the first influenza HA protein;and/or (ii) a portion of the first influenza NA protein is replaced witha homologous region from a second influenza NA protein, wherein thesecond influenza NA protein is from a different strain than the firstinfluenza NA protein.
 6. The VLP of claim 5, wherein the transmembranedomain of the first HA or first NA protein is replaced with thetransmembrane domain of the second HA or second NA protein.
 7. The VLPof claim 6, wherein the cytoplasmic tail region of the first HA or firstNA protein is replaced with the cytoplasmic tail region of the second HAor NA protein.
 8. The VLP of claim 7, wherein the second HA and NAproteins are derived from influenza virus A/PR/8/34 or A/Udorn/72.
 9. Ahost cell comprising a VLP according to claim
 1. 10. A packaging cellfor producing a VLP according to claim 1, the packaging cell stablytransfected with one or more polynucleotides encoding less than all ofthe M1, M2, HA and NA of the VLP, and further wherein upon introductionand expression of the one or more influenza protein-encoding sequencesnot stably transfected into the cell, the VLP is produced by the cell.11. The packaging cell line of claim 10, wherein a polynucleotideencoding M1 is stably transfected into the cell.
 12. The cell of claim10, wherein the cell is a mammalian cell line.
 13. An immunogeniccomposition comprising the VLP of claim 1 and a pharmaceuticallyacceptable excipient.
 14. The immunogenic composition of claim 13,further comprising an adjuvant.
 15. A method of producing a VLPaccording to claim 1, the method comprising the steps of: expressing oneor more polynucleotides encoding the M1 and influenza glycoproteins ofthe VLP in a suitable host cell under conditions such that the VLPsassemble in the host cell; and isolating the assembled VLPs from thehost cell.
 16. The method of claim 15, wherein the host cell is selectedfrom the group consisting of a mammalian cell, an insect cell, a yeastcell and a fungal cell.
 17. The method of claim 15, wherein the one ormore polynucleotides are expressed from an expression vector.
 18. Themethod of claim 17, wherein the polynucleotides are operably linked tocontrol elements compatible with expression in the selected host cell.19. The method of claim 18, wherein the expression vector is selectedfrom the group consisting of a plasmid, a viral vector, a baculovirusvector and a non-viral vector.
 20. The method of claim 15, wherein oneor more of the polynucleotides are stably integrated into the host cell.21. A method of generating an immune response in a subject to one ormore influenza viruses, the method comprising the step of administeringa composition comprising a VLP according to claim 1 to the subject. 22.The method of claim 21, wherein the composition is administeredintranasally.
 23. The method of claim 21, wherein the composition isadministered in a multiple dose schedule.
 24. The method of claim 21,wherein an immune response to more than one influenza virus strain isgenerated.